All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Hyperthermophilic organisms, i.e. organisms which thrive on temperatures around the boiling point of water (which may be significantly above 100° C. at high pressures), are found primarily in the depths of the ocean close to geothermal springs, but also occur in many other natural and artificial environments. Since these bacteria live in high temperature environments, their enzymes, which are essential to sustaining life processes such as digestion and respiration, must be able to function at such extreme temperature conditions. Enzymes in conunon mesophilic bacteria (i.e., organisms that can grow at intermediate temperatures compared to the upper and lower extremes for all organisms) typically fail at these high temperatures.
A number of organisms have been isolated from extreme environments. These organisms have been studied and certain useful compounds have been identified. For example, thermostable DNA polymerases have been obtained from Thermus aquaticus. Proteases have been isolated from thermophiles including T. aquaticus, Desulfurococcus species, Pyrococcus furiosus, Sulfolobus acidocaldarius, Thermococcus stetteri, Thermus thermophilus, and Pyrobaculum aerophilum. However, difficulties in culturing extremophiles have limited the number of these microbes which have been characterized as well as the number of useful compounds isolated therefrom (Brennan, Chemical and Engineering News, Oct. 14, 1996). The new practice of metagenome cloning may circumvent these culturing issues in the future (Rhee, J. K. et al. “New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library”, Appl. Environ. Microbiol. 71 (2): 817-825).
Stetter, et al. identified microorganisms from the hot springs of Vulcano Island, Italy, that flourish at temperatures exceeding 100° C. (Stetter, K. O. “Microbial Life in Hyperthermal Environments,” ASM News 61:285-290, 1995; Stetter, K. O., Fiala, G., Huber, R. And Segerer, A. “Hyperthermophilic Microorganisms,” FEMS Microbiol. Rev. 75:117-124, 1990). While thermophilic organisms that grow optimally at 60° C. have been known for many years, the hyperthermophilic (or extremely thermophilic) microorganisms belong to a new evolutionary class called Archaea (Woese, C. R., Kandler, O. and Wheelis, M. L. “Towards a Natural System of Organisms: Proposal for the Domains Archaea, Bacteria, and Eucarya,” Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). The Archaea are believed to have originated over a billion years ago during the epoch when the Earth was cooling. Consequently their evolutionary development was set in motion within the environment of hot springs and deep sea hydrothermal vents. One member of this new group is Pyrococcus furiosus. 
Hyperthermophilic organisms such as Pyrococcus furiosus have evolved to survive at significantly elevated temperatures. To accomplish this, the bacterial proteins, particularly those expressed in the periplasmic space (where the contact with the environment is most felt), must be well-adapted to these conditions. Pyrococcus furiosus is an obligate heterotroph that can be grown on polymeric substrates including protein and starch at temperatures of up to about 103° C. Preparations containing proteolytic enzymes prepared from Pyrococcus furiosus have been previously described in U.S. Pat. Nos. 5,242,817 and 5,391,489. See also, for example, Blumentals, Ilse I., Robinson, Anne S., and Kelly, Robert M., “Characterization of Sodium Dodecyl Sulfate-Resistant Proteolytic Activity in the Hyperthermophilic Archaebacterium Pyrococcus furiosus.” Applied and Environmental Microbiology, 56, 7:1992-1998, (1990); Eggen, Rik, Geerling, Ans, Watts, Jennifer and de Vos, Willem M., “Characterization of pyrolysin, a hyperthermoactive serine protease from the archaebacterium Pyrococcus furiosus.” FEMS Microbiology Letters, 71:17-20 (1990); Voorhorst, Wilfried G. B., Eggen, Rik I. L., Geerling, Ans C. M., Platteeuw, Christ, Siezen, Roland J., de Vos, Willem M., “Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus.” Journal of Biological Chemistry, 271, 34: 20426-20431 (1996).
In the past several years, there have been several thermophilic eukaryotic organisms discovered as well, e.g. the Pompeii worm Alvinella pompejana, which thrives in a temperature gradient of 20-80° C., in a very low pH and high heavy metal environment. Another thermo-acidophilic species, Alicyclobacillus acidocaldarius, has evolved to live at elevated temperatures and extremely low pH. For this reason, it is thought that proteins from this organism (particularly periplasmic proteins, exposed to the full brunt of extreme environmental conditions) should be able to function as sensors in such environments, which might be seen locally in such parts of the cell as the vacuole.
Concurrent to the discovery of the ever increasing number of hyperthernophilic species in recent years, in vivo measurement of ions and metabolites by using Fluorescence Resonance Energy Transfer (FRET) has been successfully used. For instance, the FRET technology has been used to measure calcium concentration changes, by fusing CFP, YFP, and a recognition domain consisting of calmodulin and the M13 peptide (Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002a) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906-918; Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002b) Creating new fluorescent probes for cell biology. Nature Reviews Molecular Cell Biology 3, 906-918). Binding of calcium to calmodulin causes global structural rearrangement of the chimera resulting in a change in FRET intensity as mediated by the donor and acceptor fluorescent moieties. Recently a number of bacterial periplasmic binding proteins, which undergo a Venus flytrap-like closure of two lobes upon substrate binding, have been successfully used as the scaffold for metabolite nanosensors (Fehr, M., Frommer, W. B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. U S A 99, 9846-9851; Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127-19133; Lager, I., Fehr, M., Frommer, W. B., and Lalonde, S. (2003) Development of a fluorescent nanosensor for ribose. FEBS Lett 553, 85-89).
Although FRET biosensors have proved to be indispensable tools in the study of metabolite levels in the living organisms, the construction of the biosensors with mesophilic ligand binding proteins has its limits. One of the limits is that the ligand binding proteins may become destabilized once mutations are introduced into the protein via protein engineering, e.g. to alter ligand-binding specificity or improve the sensor signal. For instance, a putative lactate-binding protein Lac.G2 (Looger et al., Nature, 423 (6936): 185-190) was constructed by mutation of FLIP-mglB.Ec, and although it showed evidence of lactate binding, it was significantly destabilized, as evidenced by the temperature-dependent decrease in FRET signal. It is therefore necessary to develop environmentally stable biosensors with improved thermo-, chemo-, and acido-stablity, which may provide more robust scaffolds for protein engineering.
Therefore, a need exists for improved environmentally stable biosensors which can be easily produced.